Functional identification of an FMRFamide-related peptide gene on diapause induction of the migratory locust, Locusta migratoria L.

FMRFamide-related peptides (FaRPs) are a type of neuropeptide, which participate in a variety of physiological processes in insects. Previous study showed that myosuppressin, being a member of FaRPs, initiated pupal diapause in Mamestra brassicae. We presumed that FaRPs genes might play a critical role in photoperiodic diapause induction of L. migratoria. To verify our hypothesis, flrf, a precursor gene of FaRP from L. migratoria, was initially cloned under long and short photoperiods that encoded by flrf gene identified from central nervous system (CNS). Phylogenetic analysis showed that the protein encoded by L. migratoria flrf gene, clustered together with Nilaparvata lugens (Hemiptera: Delphacidae) with 100% bootstrap support, was basically an FMRFamide precursor homologue. We noticed the availability of -RFamide peptides (GSERNFLRFa, DRNFIRFa) under short photoperiod only, which suggested their functions related to photoperiodic diapause induction. RNAi and quantitative real-time PCR (qRT-PCR) results further confirmed that the flrf gene promoted locust's diapause.

Comparative Transcriptomic Analysis Reveals Molecular Profiles of Central Nervous System in Maternal Diapause Induction of Locusta migratoria.

Egg diapause in Locusta migratoria L. (Orthoptera: Acridoidea) is believed to be influenced by maternal photoperiod. However, the molecular mechanism regulating the phenomenon of maternal diapause induction is unclear. Here we performed transcriptomic analyses from the central nervous system (CNS) of migratory locusts under long and short photoperiods to identify differentially expressed genes (DEGs) related to diapause induction. There were total of 165750 unigenes from 569491 transcripts, and 610 DEGs were obtained in S_CNS (CNS of short photoperiod treated locusts) vs. L_CNS (CNS of long photoperiod treated locusts). Of these, 360 were up-regulated, 250 were down-regulated, and 84 DEGs were found to be related to FOXO signaling pathways, including citrate cycle/TCA cycle, glycolysis/ gluconeogenesis, oxidative phosphorylation, and PI3K-Akt. The qRT-PCR validation of mRNA expression of 12 randomly selected DEGs showed consistency with transcriptome analysis. Furthermore, the takeout gene thought to be involved in circadian rhythm was cloned and used for RNAi to observe its function in maternal diapause induction. We found that the mRNA level of Lm-takeout was significantly lower in dstakeout treatments as compared to the control under both long and short photoperiods. Similarly, the offspring diapause rate was significantly higher in dstakeout treatment as compared to the control only in short photoperiod. This shows that the Lm-takeout gene might be involved in the inhibition of maternal diapause induction of L. migratoria under short photoperiods. The present study provides extensive data of the CNS transcriptome and particular insights into the molecular mechanisms of maternal effects on egg diapause of L. migratoria As well for the future, the researchers can explore other factors and genes that may promote diapause in insect species.

Molecular identification and diapause-related functional characterization of a novel dual-specificity kinase gene, MPKL, in Locusta migratoria.

Diapause is an important overwintering strategy enabling Locusta migratoria to survive under stressed conditions. We identified a novel dual-specificity kinase gene that is differentially expressed between long and short day-treated L. migratoria. To determine its function on photoperiodic diapause induction, we cloned the specific gene. Interestingly, phylogenetic analysis shows that this dual-specificity kinase is of the mycetozoa protein kinase-like (MPKL) type and may have been transferred horizontally from Mycetozoa to L. migratoria. RNA interference results confirm that MPKL promotes photoperiodic diapause induction of L. migratoria. Furthermore, MPKL significantly inhibits Akt and FOXO (i.e. forkhead box protein O) phosphorylation levels in ovaries, and also enhances reactive oxygen species, superoxide dismutase and catalase activities, whereas peroxidase activity is decreased under both photoperiodic regimes. The findings of the present study offer insight into the molecular mechanism responsible for dual-specificity kinase-induced diapause in insects.

Transcriptome Sequencing Reveals Potential Mechanisms of the Maternal Effect on Egg Diapause Induction of Locusta migratoria.

Photoperiod is one of the most important maternal factors with an impact on the offspring diapause induction of Locusta migratoria. Previous studies have shown that forkhead box protein O (FOXO) plays an important role in regulating insect diapause, but how photoperiod stimulates maternal migratory locusts to regulate the next generation of egg diapause through the FOXO signaling pathway still needs to be addressed. In this study, the transcriptomes of ovaries and fat bodies of adult locusts under a long and short photoperiod were obtained. Among the total of 137 differentially expressed genes (DEGs) in both ovaries and fat bodies, 71 DEGs involved in FOXO signaling pathways might be closely related to diapause induction. 24 key DEGs were selected and their expression profiles were confirmed to be consistent with the transcriptome results using qRT-PCR. RNA interference was then performed to verify the function of retinoic acid induced protein gene (rai1) and foxo. Egg diapause rates were significantly increased by RNAi maternal locusts rai1 gene under short photoperiods. However, the egg diapause rates were significantly decreased by knock down of the foxo gene in the maternal locusts under a short photoperiod. In addition, reactive oxygen species (ROS) and superoxide dismutase (SOD) activities were promoted by RNAi rai1. We identified the candidate genes related to the FOXO pathway, and verified the diapause regulation function of rai1 and foxo under a short photoperiod only. In the future, the researchers can work in the area to explore other factors and genes that can promote diapause induction under a long photoperiod.